Structure-function analysis of the 5' end of yeast U1 snRNA highlights genetic interactions with the Msl5*Mud2 branchpoint-binding complex and other spliceosome assembly factors.

Schwer B, Chang J, Shuman S - Nucleic Acids Res. (2013)

Bottom Line:
Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential.We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2.Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.

ABSTRACTYeast pre-mRNA splicing initiates via formation of a complex comprising U1 snRNP bound at the 5' splice site (5'SS) and the Msl5•Mud2 heterodimer engaged at the branchpoint (BP). Here, we present a mutational analysis of the U1 snRNA, which shows that although enlarging the 5' leader between the TMG cap and the (3)ACUUAC(8) motif that anneals to the 5'SS is tolerated, there are tight constraints on the downstream spacer between (3)ACUUAC(8) and helix 1 of the U1 fold. We exploit U1 alleles with 5' extensions, variations in the (3)ACUUAC(8) motif, downstream mutations and a longer helix 1 to discover new intra-snRNP synergies with U1 subunits Nam8 and Mud1 and the trimethylguanosine (TMG) cap. We describe novel mutations in U1 snRNA that bypass the essentiality of the DEAD-box protein Prp28. Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential. We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2. Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.

gkt490-F5: Effects of mutations in the 3ACUUAC8 sequence. (A) The base-pairing interaction between the U1 snRNA 3ACUUAC8 sequence and the consensus pre-mRNA 5′SS is shown. U1Δ p(CEN LEU2 U1) cells bearing the indicated U1 alleles were spot-tested for growth at the indicated temperatures. (B) Yeast strains bearing U1 WT, U5A or U5C alleles in the indicated genetic backgrounds were spot-tested for growth at the temperatures specified. Synthetic lethal interactions of U5A and U5C are indicated at bottom.

Mentions:
The base-pairing interaction between the 5′ end of yeast U1 snRNA and the consensus 5′SS of yeast pre-mRNAs is shown in Figure 5. Early studies of the effects of nucleobase changes within this segment of yeast U1 RNA identified the following lethal pyrimidine-to-purine mutations: C4A, C4G, C8A and C8G (36,37). Pyrimidine-to-pyrimidine mutants C4U and C8U were viable but slow growing at 30°C (36). The U5A allele, which eliminates a U:U mismatch in the U1•5′SS duplex (Figure 5), was also viable but slow growing at 30°C (36). Here, we examined the effects of these known viable mutations, and of several novel nucleobase changes in the 3ACUUAC8 sequence, on the activity and genetic interactions of the U1 snRNA. The C8U mutant barely grew at 37 and 34°C and failed to grow at 25, 20 or 18°C (Figure 5A). The C4U strain grew best at 34°C and failed to grow at 25, 20 or 18°C (Figure 5A). We found that C4U was lethal at 34°C in combination with tgs1Δ, cbc2-Y24A, nam8Δ, mud1Δ, swt21Δ and mud2Δ. A new allele, U1-A7G, was viable but slow growing at 25, 30 and 34°C, but did not thrive at higher or lower temperatures (Figure 5A). A7G was inviable in combination with tgs1Δ, cbc2-Y24A, nam8Δ, mud1Δ, swt21Δ and mud2Δ.Figure 5.

gkt490-F5: Effects of mutations in the 3ACUUAC8 sequence. (A) The base-pairing interaction between the U1 snRNA 3ACUUAC8 sequence and the consensus pre-mRNA 5′SS is shown. U1Δ p(CEN LEU2 U1) cells bearing the indicated U1 alleles were spot-tested for growth at the indicated temperatures. (B) Yeast strains bearing U1 WT, U5A or U5C alleles in the indicated genetic backgrounds were spot-tested for growth at the temperatures specified. Synthetic lethal interactions of U5A and U5C are indicated at bottom.

Mentions:
The base-pairing interaction between the 5′ end of yeast U1 snRNA and the consensus 5′SS of yeast pre-mRNAs is shown in Figure 5. Early studies of the effects of nucleobase changes within this segment of yeast U1 RNA identified the following lethal pyrimidine-to-purine mutations: C4A, C4G, C8A and C8G (36,37). Pyrimidine-to-pyrimidine mutants C4U and C8U were viable but slow growing at 30°C (36). The U5A allele, which eliminates a U:U mismatch in the U1•5′SS duplex (Figure 5), was also viable but slow growing at 30°C (36). Here, we examined the effects of these known viable mutations, and of several novel nucleobase changes in the 3ACUUAC8 sequence, on the activity and genetic interactions of the U1 snRNA. The C8U mutant barely grew at 37 and 34°C and failed to grow at 25, 20 or 18°C (Figure 5A). The C4U strain grew best at 34°C and failed to grow at 25, 20 or 18°C (Figure 5A). We found that C4U was lethal at 34°C in combination with tgs1Δ, cbc2-Y24A, nam8Δ, mud1Δ, swt21Δ and mud2Δ. A new allele, U1-A7G, was viable but slow growing at 25, 30 and 34°C, but did not thrive at higher or lower temperatures (Figure 5A). A7G was inviable in combination with tgs1Δ, cbc2-Y24A, nam8Δ, mud1Δ, swt21Δ and mud2Δ.Figure 5.

Bottom Line:
Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential.We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2.Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.

ABSTRACTYeast pre-mRNA splicing initiates via formation of a complex comprising U1 snRNP bound at the 5' splice site (5'SS) and the Msl5•Mud2 heterodimer engaged at the branchpoint (BP). Here, we present a mutational analysis of the U1 snRNA, which shows that although enlarging the 5' leader between the TMG cap and the (3)ACUUAC(8) motif that anneals to the 5'SS is tolerated, there are tight constraints on the downstream spacer between (3)ACUUAC(8) and helix 1 of the U1 fold. We exploit U1 alleles with 5' extensions, variations in the (3)ACUUAC(8) motif, downstream mutations and a longer helix 1 to discover new intra-snRNP synergies with U1 subunits Nam8 and Mud1 and the trimethylguanosine (TMG) cap. We describe novel mutations in U1 snRNA that bypass the essentiality of the DEAD-box protein Prp28. Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential. We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2. Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.